A. Field of the Invention
The present invention relates to novel fluorescent proteins and nucleic acid sequences derived from the genus Branchiostoma. Specifically, the invention includes fluorescent protein compositions, methods of use, and transgenic organisms encompassing the fluorescent proteins.
B. Description of Related Art
Fluorescent proteins are used extensively as markers in many biotechnological applications. These proteins become spontaneously fluorescent due to autocatalytic chromophore generation and have been found in several aquatic species including jellyfish, coral, and sea pansies. The first fluorescent protein to be used as a research tool was green fluorescent protein (GFP), initially isolated from Aequorea victoria (jellyfish). Since the initial discovery of GFP, numerous fluorescent proteins have been generated by either altering the sequence of GFP or isolation from other bioluminescent species.
The crystal structure of GFP (Ormo, M. et al., Science 273: 1392-1395, 1996) combined with mutation experiments (Li, S., et al., J Biol. Chem. 272(45): 28545-28549, 1997) have elucidated the essential domains required for the fluorescent activity of GFP. The chromophore of GFP is the result of spontaneous cyclization and oxidation of the amino acids Ser65/Thr65, Tyr66, and Gly67. Based on the crystal structure, the GFP protein structure consists of an 11-stranded beta barrel with a coaxial helix, collectively termed the β-can, in which the chromophore forms from the central helix (Ormo, M. et al., Science 273: 1392-1395, 1996). While most fluorescent proteins identified to date share the same β-can polypeptide told, substantial differences in the chromophore structure do exist (Labas, Y. A. et al., PNAS 99(7) 4256-4261, Apr. 2, 2002).
Inherent coding underlies many limitations encountered in the use of common fluorescent proteins. Despite extensive use, the expression of fluorescent proteins in mammalian cells is highly variable at best, often requiring a strong promoter and decreased incubation temperature (Ogawa, H., et al., Proc. Natl. Acad. Sci. 92: 11899-11903, 1995). Fluorescent proteins exhibit a slow maturation rate and decreased expression efficiency in non-homologous cells, including mammalian cells. To enhance fluorescent proteins used in mammalian cells, attempts have been made to harmonize, or humanize, fluorescent proteins by replacing one or more species-specific codons with codons more frequently used in human genes to produce specific amino acids (U.S. Patent Application No. 2005/0014223A1, filed: Apr. 1, 2004; U.S. Patent Application No. 005968750A, filed: Oct. 9, 1998; Yang, T., et al., Nucleic Acids Research 24(22): 4592-4593). Harmonizing fluorescent proteins improves translation efficiency and maturation rate in host cells, resulting in better expression and brighter fluorescence. Further optimization of fluorescent protein coding sequences by modifying specific amino acids may result in altered folding properties, shifted excitation/emission spectra, and altered incubation temperature sensitivity (Nagai, T. et al., Nature Biotechnology 20(1): 87-90, January, 2002).
While fluorescent proteins are useful, the use is limited by incompatible species differences. Fluorescent proteins are not expressed or well-folded at temperatures suitable for growing mammalian cells (typically about 37′C). Modifying the amino acid sequence of GFP has produced derivatives with enhanced fluorescence in non-homologous cells at incubation temperatures above 30° C. (U.S. Patent Application No. 2004/0138420A1, filed: Jan. 14, 2005). Furthermore, amino acid substitutions corresponding to F64, either S65 or E222, and/or S175 has increased fluorescence detection in mammalian cells at lower levels of GFP expression. Nevertheless, despite numerous attempts at optimizing existing fluorescent proteins for use in mammalian cells, the use of fluorescent proteins in mammalian cells is still limited due to the inability to fully overcome these drawbacks. The fluorescent proteins of the present invention inherently possess characteristics that overcome the limitations and drawbacks faced using available fluorescent proteins of the art.